Cytokine preparation for immunotherapy

ABSTRACT

A cytokine composition is prepared for use in immunotherapy of various diseases. The composition is prepared by culturing leucocytes previously infected with a virus in a culture medium containing a transal composition isolated from plasma and a complex of proteinase inhibitors composition isolated from plasma, separating leucocytes from the medium, removing impurities from the medium, and recovering a cytokine composition containing the transal composition and the complex of protease inhibitors composition. The transal composition contains transferrin, albumin and a 40 kDa protein and the complex of protease inhibitors composition contains α 2  -macroglobulin, a 160 kDa protein, an 80-60 kDa protein and a 20 kDa protein. The transal composition is prepared by mixing plasma with chloroform and recovering an aqueous phase, precipitating gamma globulins from the aqueous phase with polyethylene glycol to recover another aqueous phase, passing this aqueous phase through an anion exchange column and recovering proteins that bind to the column. The complex of protease inhibitors composition is prepared using steps for preparing the transal composition except that additional steps are used of collecting eluate from the anion exchange column, passing the eluate through a Cu ++  chelate anion exchange column and recovering proteins that bind to the column.

FIELD OF THE INVENTION

The present invention relates to a novel cytokine composition, a methodfor its preparation, and the use of the preparation in the immunotherapyof various diseases such as bacterial infections, viral infections andcancers.

BACKGROUND OF THE INVENTION

The immune system provides defence for the body against infectiousagents such as bacteria, viruses, fungi and parasites. More recently, ithas been shown that the state of the immune system can be linked to thesusceptibility to developing certain types of cancers. For example, ithas been shown that in certain immune deficient states, most notablyAcquired Immune Deficiency Syndrome (AIDS), the incidence of cancer isgreatly increased. Immunotherapy has become an important therapy againstvarious infectious agents and even in the treatment of cancer. Onebranch of immunotherapy involves using immunomodulating agents that canmodulate, induce or increase the immune response.

One group of immunomodulating agents that has been extensively studiedincludes the cytokines. Cytokines are the chemical messengers of theimmune system and are produced by immune cells such as lymphocytes andmonocytes. One cytokine, namely interferon (IFN), has been shown notonly to induce the immune system but also to act directly on tumors toinhibit proliferation of the tumor cells. Interferon is well known inthe art. Details on its characterization as well as methods for itspreparation can be found in Methods in Enzymology, Volume 119,"Interferons, PART C" 1986.

Although both crude as well as purified preparations of cytokines, suchas interferon, are currently available, the problems with these knownpreparations include the following. Crude preparations generally containproteinases which break down the cytokines and thereby decrease thestability of the preparation. Proteinase inhibitors are available butthese are generally toxic or antigenic to man and thus are not useful intherapeutic applications. On the other hand, while purified interferonis generally proteinase free, the methods for purifying interferon knownto date generally result in a poor yield of interferon. Furthermore, thebiological activity of a purified preparation is generally lower thanthat of a crude interferon preparation.

Early in the 80s, it was observed that a crude preparation of interferonactivated human natural killer cells (NK) more effectively than purifiedIFN prepared from the same batch of crude IFN. As shown in Table A thehighest degree of NK activation can be achieved with a crude IFN titerof 50 IU/ml and a purified IFN titer of 625 IU/ml. These titers resultedin activation of the NK cells of 550% and 600%, respectively. From thisdata it was concluded that crude IFN has a higher potential ofimmunoactivation which is likely due to the presence of some othercytokines together with IFN.

It was later found that mixed cultures of donor Leukocytes produced (inresponse to viral induction and interaction of cells with differentgenotypes) cytokines as such as Interleukin-1 (IL-1), Tumor NecrosisFactor (TNF), Migration Inhibition Factor (MIF), Leucocyte MigrationInhibition Factor (LIF), together with IFN-α. It is known that thesecytokines participate in the activation of immune effector cells (mainlymacrophages, T₄ -lymphocytes, neutrophils) and participate in the immunerecognition of antigen and its elimination from the body. Thesereactions present the first stage of the immune response manifested byeffectors in response to an antigen encounter. Amplification of immuneresponse, developing specific humoral reactions and memory cells areevents that occur later and depend on effective immune reactions in thefirst (non-specific) stage of immune response.

The cytokines such as IFN, IL-1, TNF, MIF and LIF, have differentphysico-chemical properties, therefore it is very difficult to retaincomposition that is active and composed of a mixture of the cytokinespreparation after chemical purification procedures. Furthermore, formedical preparations containing protein it is very important to limitthe quantity of total protein because foreign proteins (especially whenused by parenteral routes) can induce sensitization. However, loweringof the level of protein increases the sensitivity of the active factorsto proteolysis. As a result, the activity of such preparations aftertechnological procedures is-decreased and problems with storage of thedrug forms appear.

Therefore, in view of all of the above it is desirable to produce acytokine composition that includes a mixture of cytokines and that islargely substantially free of proteinases while at the same time showinggood biological activity.

SUMMARY OF THE INVENTION

The present invention relates to a novel cytokine composition (which hasbeen termed leukinferon) as well as a method for its preparation and itsuse in the immunotherapy of various conditions. The cytokine compositionis produced by virally induced donor leucocytes. The cytokinecomposition comprises human leukocyte interferon IFN-α as well as othercytokines of the first, non-specific, phase of the immune response(I1-l, TNF, MIF, LIF, etc.) in their naturally occurring ratio.

The novel methods according to the present invention produce a cytokinecomposition that avoids the drawbacks of the prior art. One novelingredient of the cytokine composition is a complex of proteinaseinhibitors (CPI), that has previously been isolated from donor plasma.CPI inhibits proteolysis reactions in cytokine preparations.Furthermore, since CPI is naturally occurring and isolated from humans,it is non-toxic to humans.

A second ingredient, a novel protein composition, which has been termedtransal, is also added during the preparation of the cytokinecomposition according to the present invention. Transal is a naturallyoccurring group of proteins that is isolated from donor plasma. Afterthe addition of transal and CPI, the culture medium containing thecytokines is further treated to remove impurities such as human viruses,antigens and antibiotics or other low molecular weight components.

Accordingly, the present invention provides a method of producingtransal comprising the steps of:

a) mixing plasma with chloroform,

b) recovering a first aqueous phase,

c) precipitating gamma globulins from the first aqueous phase;

d) recovering a second aqueous phase;

e) passing the second aqueous phase through an ion exchange column,

f) recovering proteins that bind to the column.

The present invention also provides a transal composition comprisingtransferrin, albumin and a 40 kDa protein.

The present invention further provides a method of producing a complexof proteinase inhibitors comprising the steps of:

a) mixing plasma with chloroform,

b) recovering a first aqueous phase,

c) precipitating gamma globulins from the first aqueous phase;

d) recovering a second aqueous phase;

e) passing the second aqueous phase through an ion exchange column,

f) collecting an eluate that passes freely through the column,

g) passing said eluate through a Cu⁺⁺ chelate column,

h) recovering proteins that bind to the column.

The present invention further provides a complex of proteinaseinhibitors composition comprising α₂ -macroglobulin, a 160 kDa protein,an 80-60 kDa protein and a 20 kDa protein.

The present invention also provides a process for the production of acytokine composition comprising the steps of;

a) culturing virally-induced leucocytes with transal and complex ofproteinase inhibitors in liquid culture medium;

b) removing the leucocytes from the liquid; and

c) treating the liquid to remove impurities.

The leukinferon composition has been shown to be effective in thetreatment of a variety of disease conditions such as bacterialinfections, viral infections, as well as certain types of cancer.

DETAILED DESCRIPTION OF THE INVENTION

1. Method of Preparation of Transal

0.5 L of donor plasma is freshly collected, anticoagulant is added, andthe plasma is cooled to 6°±2° C. Chloroform is added (10% volume/volume)and the mixture is agitated for 30 minutes and then allowed to stand foranother 10 to 15 minutes to allow the upper aqueous and the lowerchloroform layers to separate. The aqueous layer is removed andcentrifuged at 2,000 x g, for 20 minutes at 6°±2° C. The supernatant isremoved and adjusted to pH 8.5 with 0.02M NaOH. Subsequently, a 1/5(v/v) solution of 30% polyethylene glycol is added and the mixture iskept at room temperature for 20 minutes. The gamma-globulin fractionprecipitates and can be separated by centrifugation at 2,000 x g, 15min. at 10°±2° C. The resulting supernatant is then passed through aDEAE cellulose column (DEAE-52Servacel, Serva, Germany) which has beenequilibrated with 0.01M tris-HCl buffer, pH 8.5 and 0.01M NaCl. Thefraction passing freely through the column is collected and used toprepare the complex of proteinase inhibitors (CPI) which will bediscussed in more detail below. The column is washed and then thefraction binding to the column (which is a fraction containing thetransal) is eluted with 0.01M tris-HCl buffer, pH 8.5 and 0.5M NaCl. Thevolume of the eluate containing the transal is, generally around 0.25liters and has a protein concentration of 5 to 15 mg/ml. Polyacrylamidegel electrophoresis of the eluate shows that the main components of theeluate are albumin and transferrin. There is also present a minorprotein with a molecular weight of 40 kDa. The entire eluate is termedtransal. The eluate is substantially or totally free of gamma globulin.Accordingly, the term "transal" as used herein denotes the product ofthe above described process which comprises a mixture of transferrin,albumin and a 40 kDa protein.

The eluate is desalted by dialysis against 0.01M Na phosphate buffer, pH7.2 with 0.15M NaCl (PBS) or by gel filtration through a Sephadex G-25"coarse" column and sterilized by 0.2 μ membrane filtration.

Table 1 is a comparative study showing the effect of transal, plasmal ornative plasma on interferon (IFN) production in a crude interferonpreparation. Plasmal is plasma that has been treated to remove gammaglobulins. This table shows that with transal, one can use 3.5 timesless protein as compared with native plasma, or 2.8 times less proteinas compared with plasmal to obtain the same yield of IFN activity.Therefore, this table shows that transal is a very effective componentin a cytokine composition.

2. Preparation of the Complex of Proteinase Inhibitors

One of the main problems with crude cytokine preparations is that theproteinase activity is generally high and causes the proteolysis of thecytokines in the composition. Many non-specific and specific inhibitorsof proteinases in crude interferon compositions were studied and it wasshown that these compositions are generally resistant to suchinhibitors. In particular, it was shown that each of dextran,polyglycine, ethylenediaminotetra-acetate, aminocaproic acid, andkontrycal in a concentration of 0.1-1.0 mg/ml had no effect onproteolysis determined by the azacasein test. Soybean trypsin inhibitorat 1 mg/ml reduced proteolysis by one half.Phenylmethylsulfonidefluoride (Serva, Germany), was found to be a highlyeffective inhibitor. However, it is very toxic which prevents it frombeing used in medical compositions.

Proteinases are present in extracellular biological fluids and they arecommon mediators of the acute phase of inflammation. The body also hasproteinase inhibitors to control the proteolysis by the proteinase. Forexample, proteinase inhibitors with wide spectrum inactivation, such asα₂ -macroglobulin, are present in healthy donor plasma in quite largequantities--up to 3 mg/ml. Therefore, it was desirable to isolate suchnaturally occurring proteinase inhibitors for use in medicalpreparations.

The naturally occurring complex of proteinase inhibitors (CPI) wasisolated from the fraction freely eluting from the DEAE Sepharose columnas described in the preparation of transal in 1) above. The eluate wascollected and the pH was adjusted to 6.5 with 0.02M HCl. The eluate wasthen loaded on to a Cu⁺⁺ chelate Sepharose 4B (Pharmacia, Sweden) columnthat had previously been equilibrated with 0.02M phosphate buffer, pH6.5 and 0.8M NaCl. The column was washed several times with a 0.02Mphosphate buffer solution at pH 7.4 with 0.08M NaCl. The inhibitors wereeluted with 0.1M acetate buffer solution, pH 4.5 and 0.5M NaCl. Thiseluate contained protein that gave two peaks, the antiproteinaseactivity being present mainly in the first peak. Material correspondingto the first peak was dialysed against 0.01M phosphate buffer solution,pH 7.2 with 0.15M NaCl. Alternatively, it may be desalted on a SephadexG-25 column (Pharmacia, Sweden), by adding 0.1M phosphate buffer pH 7.2with 1.5M HCl at 1/10 of the eluate volume.

Starting from 0.5 liters of donor plasma it is possible to obtainapproximately 264±56 ml of the proteinase-containing fraction with anaverage of 13.26±1.44 ml of total protein. The composition of CPI wasanalyzed by electrophoresis on polyacrylamide gel. The gel shows thatCPI contains 4 fractions. The major protein corresponds to the α₂macroglobulin standard. The minor fractions correspond to 160, 80-60 and20 kDa. Accordingly, the term "complex of proteinase inhibitors" (CPI)as used herein refers to the product of the above described processwhich comprises a mixture of α₂ macroglobulin, a 160, an 80-60 and a 20kDa protein.

The effect of the CPI on proteinase activity is shown in Table 2. Thelevel of proteinase activity is compared to a 0.025% trypsin insolution. As can be seen from this table, a preparation containing onlyplasmal has as much as 27% of proteinase activity as compared to thestandard. However, when using a preparation containing transal (6%) aswell as CPI (at 50 mg/ml), no proteinase activity is observed.

3. Preparation of Leukinferon

Leukocytes are isolated from buffy coats collected from healthy donorswith the use of erythrocyte precipitants, such as dextran or polyvinylalcohol and subsequent hemolysis. Leukocytes are cultured at 37.5° C. inculture medium containing insulin, human plasma, heparin and 100 IU/mLhuman IFNα. After 2 hrs incubation the inducer virus--Newcastle diseasevirus--is added and incubation is continued for 20 min. Inducedleukocytes are collected by centrifugation at 600 x g, 20 min, 6°±2° C.and are suspended in culture medium number 199 or minimal Eagles mediumwithout phosphate at a concentration of 6 million cells/mi. A 6% v/vsolution of transal as well as 15 mg/ml of CPI and 0.05M sodiumsuccinate is added to the cells which are cultured for another 10 to 14hours at 37.5° C. The cells are then separated by centrifugation at 600x g for 20 minutes at 6°±2° C. The resulting supernatant ms further usedto prepare the leukinferon.

The supernatant is treated to remove impurities such as human viruses,antigens of virus inducer and chicken allantoic fluid as well asantibiotics and other low molecular weight components of the culturemedium. For example, the antigens may be removed by negativeimmuno-absorption on antibodies which have been obtained from the serumof animals that have previously been immunized with these antigens andthen immobilized on a neutral carrier. The human viruses may beinactivated chemically by treatment with hydrogen peroxide inchloroform. The antibiotics and other low molecular weight componentsmay be removed by gel filtration through Sephadex G-25 orultrafiltration on membranes with an excluding limit of not more than 10kDa.

In one preferred embodiment, the above described impurities may beremoved as follows. The pH of the supernatant is adjusted to 8.0(0.01NaOH) and NaCl is added as a solid to final concentration 0.5M.Hydrogen peroxide is added to concentration 0.5 % (v/v) and the crudecomposition is kept at least two days at 6°±2° C. A column (150×26 mm)containing 80 mL of antibodies to viral and allantoic fluid antigens,immobilized on Sepharose 4B, is used for purification of 2.0±0.1 L ofcrude cytokine composition with a capacity 0.1 L/hr.

Before the column is used it is washed with a solution of 10 μg/μl CPIin 0.01M phosphate buffer pH 8.0 and 0.5M NaCl. CPI is also added to thecrude composition to final concentration 50 μg/ml. Then, the crudecomposition is passed through the column with velocity up to 100 ml/hr.

In the immunoabsorption process the active components of the crudepreparation pass freely through the column while the antigenicimpurities bind with antibodies and are held by the column. This method(termed negative immunoabsorbtion) allows the composition of all activefractions to remain unchanged.

The fraction passing through the column is collected and treated withchloroform to inactivate viruses. Chloroform is added at a concentrationof 10% (v/v). The mixture is kept 30 min at room temperature withagitation. The agitation is stopped for 10 min and the upper layer iscollected and centrifuged at 6000 x g, 20 min, 6°±2° C. The lower layerof chloroform extract and precipitate are discarded. The supernatantafter centrifugation is desalted by chromatography on a column (60×2800)containing 1.1 kg Sephadex G-25 "coarse" (Pharmacia, Sweden) withvelocity up to 0.5 L/hr.

The protein fraction is collected and dialyzed against 0.01M PBS, 0.15MNaCl and 1 mg/ml mannite. The supernatant is then sterilized byfiltration on 0.2 μ membrane, dispensed into ampoules and lyophylizedfor storage.

EXPERIMENTAL DATA

Leukinferon has been tested in numerous in vitro and in vivo clinicalstudies and has been shown to have very significant immunomodulatoryactivity as exemplified below.

IN-VITRO STUDIES EFFECT ON MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)EXPRESSION

It is well known that MHC antigens play a key role in immune responsesand they take part directly in the immune recognition of foreignantigen. It is also known that MHC expression is stimulated by IFN-γwhile IFN-α has little effect, in vitro, on MHC expression.

The effect of leukinferon on the MHC expression of Human LeukocyteAntigen (HLA-DR) on mononuclear cells from donor blood was compared withpurified alpha interferon (IFN-α) as well as gamma interferon (IFN-γ).The results are shown in Table 3. As can be seen from Table 3,leukinferon showed an index of amplification of 4.4 compared to 3.1 forgamma interferon and 1.4 for alpha interferon.

B. EFFECT ON HIV INFECTION

Leukinferon was tested for its ability to inhibition infection of MT4cells by the Human Immunodificiency Virus (HIV), in vitro. The resultsare summarized in Table 4. As can be seen from this Table, leukinferonat concentrations as low as 5U/ml inhibited the infection of MT4 cellsby HIV-1 even after 7 days in culture.

CLINICAL TRIALS

The leukinferon preparation has been tested on a number of patients withbacterial infections, vital infections and cancer. Some of the diseasestested to date are summarized in Table 5.

The results of the clinical studies will be described in detail withregards to four specific examples, namely sepsis, salpingoophorite,hepatitis B and breast cancer.

EXAMPLE 1 Sepsis

Eighteen young children suffering from sepsis caused by gram-negativemicroorganisms were given 4-6 intramuscular injections of theleukinferon every second day in combination with basic therapy such asantibiotics, analgesics. After treatment, various parameters ofimmunological function were assayed. The results are shown in Table 6.As can be seen from Table 6, when leukinferon was administered alongwith the basic therapy, the number of circulating immune complexes (CIC)was reduced, the killing function of neutrophils was restored (asdetermined by NCT test) as was the number of phagocytic neutrophils. Thephagocytic index also was increased when calculated as the number ofbacteria ingested. The completeness of phagocytosis also increased.

These results indicate that administration of leukinferon enhance theeffectiveness of the immune system. Clinical improvement was also fasterwith the group treated with leukinferon.

EXAMPLE 2 Salpingoophorite

Salpingoophorite is an inflammation of the tuba uterina caused by amixed bacterial infection of E. coli, Klebsiella, Candida, etc. Thiscondition generally results in infertility. Thirty women suffering fromsalpingoophorite were given, intramuscularly, one ampoule of leukinferontwice a day for three days and then one ampoule a day for two days.Blood samples were taken before treatment as well as 5 to 7 daysthereafter. The results of the effect of leukinferon on the blood cellsof the women are shown in Table 7. As can be seen from Table 7,leukinferon therapy has no effect on the levels of immunoglobulin M or Gor on the level of circulating C3-C4 components of complement. Thenumber of differentiated B-lymphocytes increased but not significantly.However, the numbers of T-lymphocytes increased significantly as didtheir functional activity as shown by RBTL on PHA and E-RFC.

Following treatment, the recovery rate was 60% and full restoration ofchild bearing function was observed in 41% of the women.

EXAMPLE 3 Breast Cancer

Leukinferon was administered by adoptive immunotherapy to womensuffering from Stage III Breast Cancer. The women's leukocytes werecollected by cytophoresis, incubated with leukinferon and transfusedback to the patient. These mononuclear cell populations were assayedfollowing treatment. The results are also shown in Table 8. As can beseen from this Table, leukinferon therapy resulted in an increase in thenumber of cells expressing MHC, an increase in T-helper, NK and othercytotoxic sub-populations having the markers CD4, CD11ab and CD38.However, the quantity of T-suppressor lymphocytes (CD8, CD22 and CD58)did not change, resulting in an increased ratio of T4/T8 cells. Thelevel of circulating monocytesmyeloid cells (CD15) was slightly reduced.

The women were observed for a period of 2 years and on average cancerwas decreased by almost three times.

EXAMPLE 4 Acute Hepatitis B

Leukinferon was administered to patients with acute hepatitis B. Theresults of this study demonstrated that leukinferon induced the fastreverse development of the main clinical symptoms of infection andintensified the normalization of laboratory data characterized by theliver function. Replication of the hepatitis virus in the body wascompletely inhibited in most patients. The immunostimulating activity ofleukinferon was also observed as the number of T-lymphocytes wasincreased, natural killer cells were activated, the frequency of seroconversions of surface and internal antigens of the virus wereincreased. After six months, it appeared the viral surface antigen inpatients treated with leukinferon was completely absent as compared to30% presence in patients that received a placebo.

In summary the above examples illustrate that leukinferon has a positiveeffect on the immune system which, as illustrated herein and has beenshown extensively in the literature, generally results in a positiveprognosis for patients suffering from diseases in which the immunesystem is somehow depressed or is required in order to effectivelycombat the infection or disease.

Examples of Treatment Protocols

While examples 1-4 illustrate the results of clinical studies done onfour specific illnesses, as previously stated, leukinferon has beentested on a wide variety of bacterial and viral infections as well asdifferent forms of cancer. While the treatment regime varies dependingon the disease or illness, some guidelines some guidelines are givenbelow regarding the use of interferon in different situations.

Recommended Courses for Bacterial Infections Prophylaxis

LF is highly effective for prophylaxis against bacterial complicationsin surgical patients with functional immunodeficiency. For this purpose,LF may be used, via intra-muscular (IM) injections, 1-2 times everyother day before surgery, on the next day after and then 2-3 injectionsevery other day. Such a regime can reduce the frequency of postsurgicalcomplications and promote the healing processes of the wound.

For prophylaxis of postnatal infections in women following childbirth,LF can be given IM on the day of delivery and 1-2 times every other daythereafter. Indications are inflammatory phenomena in placenta,pyelonephritis and other bacterial diseases.

In serious situations when medical help is not readily available,patients may be given LF IM daily or every other day to preventbacterial infection.

Therapy

In acute bacterial infections, LF may be given IM daily or every otherday (up to 5 injections) at the beginning of the therapy and then 2times a week until evidence of stabilization of immune status andclinical symptoms disappear.

In the therapy of lung diseases, IM injections may be combined withaerosol inhalations of 1-2 ampoules of LF dissolved in 5-10ml 1-2 timesa week.

In patients with purulent peritonitis, including pelvioperitonitis, LFmay be given as 3 injections IM every other day and then twice a week.For such patients, an effective combination is IM injections combinedwith local introduction of 2 ampoules of LF dissolved in 50 ml of salinewith antibiotics for treatment of the abdominal cavity.

Patients with pyelonephritis may be given LF, 2-3 I.M. injections everyother day and then twice a week for 1-3 weeks.

Viral Infections

For therapy of acute hepatitis B, LF may be injected 1-3 times a day forfirst 1-3 days (intensity depends on gravity of intoxication phenomena),then every other or third day up to occurrence of stable normalizationof liver function and suppression of antigenemia.

In patients with chronic hepatitis B, LF may be give IM 2-3 injections aweek.

Patients with influenza or other acute respiratory viral infections, LFmay be give via IM injections combined with inhalation of one ampoule,for 3 days by each route.

In therapy of shingles and chicken-pox, it is recommended to use LFdaily for 3 days, then every other day. It is useful to apply LF locallyby irrigating the oral cavity (one ampoule dissolved in 5-10 ml ofsaline). The same course is effective in treating viral stomatitis.

Oncological diseases

In patients with basal cell carcinoma, LF may be injected SC 2 times aday in healthy skin bordering the lesions.

To accelerate the healing processes and retard the metastasesdevelopment in patients with breast cancers and sarcoma, LF may be given1-3 injections IM before surgery and 1-3 times every day after and then2 injections a week.

To improve antitumour effectiveness of the therapy it is recommended tocombine LF with human leucocyte interferon for injections of 1×10⁶ IU1-2 times a week.

                  TABLE A                                                         ______________________________________                                        ACTIVATION OF HUMAN NK BY CRUDE AND                                           PURIFIED IFN PREPARED FROM THE SAME                                           BATCH OF LEUCOCYTES                                                                         IFN activity                                                    Preparations  (IU/ml)    NK activation.sup.X                                  ______________________________________                                        Crude IFN-α                                                                            10        430%                                                                50        550%                                                               250        480%                                                 Control 1                 20%                                                 Purified IFN-α                                                                         25        220%                                                               125        250%                                                               625        600%                                                 Control 2                0                                                    ______________________________________                                         .sup.X Lymphocytes were isolated from buffy coats by sedimentation from       ficollverografine solution (D = 1,077). Target cells  K562  cultivated in     monolayer in RPMI1640 with 10% fetal serum, glutamine and antibiotics.        Before use the target cells were incubated with 50 μCu Na.sup.51           CrO.sub.4 per 7 mln cells. Radioactivity was measured in cultural fluid i     a 16 hour cytotoxic test.                                                     Control 1  medium after cultivation of noninduced cells.                      Control 2  protein fraction purified from placebo by method known for         Human Leucocyte Interferon.                                              

                  TABLE 1                                                         ______________________________________                                        EFFECT OF VARIOUS PROTEIN                                                     PREPARATIONS ON IFN-α ACTIVITY.sup.x                                                 The level of Total                                                                         Yield activity                                                   Protein after                                                                              IFN-α (IU on                                  Protein components                                                                         Biosynthesis 1 × 10.sup.3                                  added to medium                                                                            (mg/ml)      leukocytes)                                         ______________________________________                                        Donor plasma (4%)                                                                          3.0 ± 0.3 2.67                                                Plasmal (4%) 2.4 ± 0.4 2.67                                                Transal (6%) 0.85 ± 0.13                                                                             2.67                                                ______________________________________                                         .sup.x data from 3 experiments                                           

                  TABLE 2                                                         ______________________________________                                        ACTIVITY OF PROTEINASES PRESENT IN                                            CRUDE IFN-.sup.x                                                                                Activity or proteinases.sup.xx                                                                 Calculated                                                                    in % of                                                                       standard                                                           Extinction trypsin                                    Preparations                                                                             Protein (mg/ml)                                                                            at 366 nm  solution                                   ______________________________________                                        Trypsin    --           0.4        100                                        Standard                                                                      0.025%                                                                        Plasmal    2.3 ± 0.4 0.11 ± 0.01                                                                            27                                        Transal + CPI                                                                            0.67 ± 0.10                                                                             0           0                                         ______________________________________                                         .sup.x data of 3 experiments                                                  .sup.xx activity of proteinases is determined by azacasein test          

                  TABLE 3                                                         ______________________________________                                        THE INFLUENCE OF DIFFERENT IFN                                                PREPARATIONS ON HLA-DR EXPRESSION                                             OF MONONUCLEAR CELLS OF DONOR BLOOD                                                           Concentration                                                                             Index                                             Preparations    (IU/ml)     amplification                                     ______________________________________                                        IFN-α     100         3.1 ± 1.0                                      Leukinferon     100         4.4 ± 2.0                                      IFN-α (recombinant)                                                                     100         1.4 ± 0.5                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                                     Day                                                              Leukinferon Conc.                                                                            3        4     5     6    7                                    ______________________________________                                         0.5 u/ml                                                                              MT4-HIV.sup.1                                                                           .sup. N.D..sup.3                                                                       --  N.D.  ++   ++                                          MT4.sup.2 N.D.     --  N.D.  --   --                                  5.0 u/ml                                                                              MT4-HIV   N.D.     --  N.D.  --   --                                          MT4       N.D.     --  N.D.  --   --                                  25.0 u/ml                                                                             MT4-HIV   --       --  --    --   N.D.                                        MT4       --       --  --    --   N.D.                               100.0 u/ml                                                                             MT4-HIV   --       --  --    --   N.D.                                        MT4       --       --  --    --   N.D.                               ______________________________________                                         .sup.1 MT4 cells incubated with HIV                                           .sup.2 MT4 cells alone                                                        .sup.3 Not Done                                                          

                  TABLE 5                                                         ______________________________________                                        LIST OF CONDITIONS IN WHICH CLINICAL TRIALS                                   HAVE BEEN CONDUCTED USING LEUKINFERON                                         ______________________________________                                        A. BACTERIAL INFECTIONS:                                                      Prophylaxis                                                                   Infectious complications after surgery or child                               birth.                                                                        Therapy                                                                       Purulent-septic infections                                                    Acute and chronic bronchitis                                                  Acute and chronic pneumonia                                                   Legionairres disease                                                          Pulmonary tuberculosis                                                        Salmonellosis                                                                 Acute and chronic pyelonephritis                                              Peritonitis                                                                   Salpingo-ophoritis                                                            B. VIRAL INFECTIONS                                                           Acute and chronic viral hepatitis                                             Influenza and other viral respiratory infections                              Shingles                                                                      Chicken-pox                                                                   Stomatitis                                                                    C. ONCOLOGICAL DISEASE                                                        Basal cell carcinoma                                                          Breast Cancer                                                                 ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        THE EFFECT OF LEUKINFERON THERAPY ON                                          VARIOUS IMMUNOLOGICAL PARAMETERS IN                                           YOUNG CHILDREN WITH SEPSIS                                                    Immunological parameters                                                                             Spon- Phago-                                                                              Phago-                                                                              Complete-                                                   taneous                                                                             cytic cytic ness of                                                     NCT-  neutro-                                                                             index phago-                                      IRL     CIC     test  philis                                                                              (cell/                                                                              cytosis                              Groups (u/ml)  (u/ml)  (%)   (%)   lymf) (%)                                  ______________________________________                                        Basic  3.0 ±                                                                              221 ±                                                                              8.5 ±                                                                            31.4 ±                                                                           3.9 ±                                                                            38.4                                 therapy                                                                              0.3     14      0.9   4.9   0.7                                        + Leu- 25.2 ±                                                                             70 ± 27.7 ±                                                                           62.8 ±                                                                           7.2 ±                                                                            88.5                                 kinferon                                                                             5.4     12      6.2   9.2   1.1                                        ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        STUDY OF VARIOUS IMMUNOLOGICAL                                                PARAMETERS IN PATIENTS WITH                                                   SALPINGOOOPHORITE FOLLOWING TREATMENT                                         WITH LEUKINFERON (30 PATIENTS).                                               Immunological                                                                 Parameter   Before therapy                                                                            After therapy                                                                            Reliability                                ______________________________________                                        IgM (g/l)   1.3 ± 0.1                                                                              1.3 ± 0.1                                                                             not                                        IgG (g/l)   8.9 ± 0.5                                                                              9.4 ± 0.3                                                                             not                                        T.sub.total 624 ± 32 938 ± 31                                                                              p<0.001                                    RBTL on PHA 24.2 ± 1.7                                                                             29.1 ± 1.7                                                                            p<0.001                                    E-RFC       23.4 ± 1.8                                                                             30.0 ± 1.5                                                                            p<0.001                                    B.sub.total 496 ± 38 558 ± 20                                                                              not                                        Circulating immune                                                                        3.5 ± 0.3                                                                              2.6 ± 0.2                                                                             0.01                                       complexes                                                                     C.sub.3 (g/l)                                                                             0.98        1.0        not                                        C.sub.4 (g/l)                                                                             0.24        0.24       not                                        ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        THE INFLUENCE OF LEUKINFERON OF DYNAMICS                                      OF MONONUCLEAR SUBPOPULATIONS IN PATIENTS                                     WITH BREAST CANCER                                                            Subpopulation     Antigens of differentation                                  ______________________________________                                        INCREASED:                                                                    T-helpers, NK and other                                                                         (CD4, CD11a, CD11b, CD38)                                   cytotoxic subpopulations                                                      The level of activated mono-                                                                    (HLA-A,B,C, HLA-DR)                                         nuclears, expressing MHC                                                      antigens                                                                      WITHOUT CHANGES OR                                                            DECREASED:                                                                    T-supressors      (CD8, CD58, CD22)                                           DECREASED:                                                                    The level of monocyte-myeloid                                                                   (CD15)                                                      cells                                                                         ______________________________________                                    

I claim:
 1. A method of producing a transal composition containingtransferrin, albumin and a 40 kDa protein, said method comprising thesteps of:a) mixing plasma with chloroform; b) recovering a first aqueousphase from the plasma/chloroform mixture of step a) and adjusting the pHof the first aqueous phase to 8.5; c) precipitating gamma globulins fromthe first aqueous phase with polyethylene glycol; d) recovering a secondaqueous phase from step c); e) passing the second aqueous phase throughan anion exchange column equilibrated with 0.01M tris-HCl buffer, pH 8.5and 0.01M NaCl; and f) recovering proteins that bind to the column bypassing a solution of 0.01M tris-HCl buffer, pH 8.5 and 0.5M NaClthrough the column, wherein the proteins eluted in step f) consist ofthe transal composition.
 2. A transal composition prepared according tothe method of claim
 1. 3. A transal composition isolated from plasmaconsisting essentially of transferrin, albumin and a 40 kDa protein. 4.A method of producing a complex of proteinase inhibitors compositioncontaining α₂ -macroglobulin, a 160 kDa protein, an 80-60 kDa proteinand a 20 kDa protein, said method comprising the steps of:a) mixingplasma with chloroform; b) recovering a first aqueous phase from theplasma/chloroform mixture of step a) and adjusting the pH of the firstaqueous phase to 8.5; c) precipitating gamma globulins from the firstaqueous phase with polyethylene glycol; d) recovering a second aqueousphase from step c); e) passing the second aqueous phase through an anionexchange column equilibrated with 0.01M tris-HCl buffer, pH 8.5 and0.01M NaCl; f) collecting an eluate that passes freely through thecolumn and adjusting the pH of said eluate to pH 6.5; g) passing saideluate through a Cu⁺⁺ chelate anion-exchange column equilibrated with0.02M phosphate buffer, pH 6.5 and 0.8M NaCl; and h) recovering proteinsthat bind to the column by eluting said column with 0.1M acetate buffer,pH 4.5 and 0.5M NaCl wherein the proteins eluted in step h) consist ofthe complex of proteinase inhibitors composition.
 5. A complex ofproteinase inhibitors composition prepared according to claim
 4. 6. Acomplex of proteinase inhibitors composition isolated from plasmaconsisting essentially of α₂ macroglobulin, a 160 kDa protein, an 80-60kDa protein and a 20 kDa protein.
 7. A method for producing a cytokinecomposition comprising the steps of:a) culturing leucocytes previouslyincubated with a virus in a liquid medium containing a transalcomposition isolated form plasma consisting essentially of transferrin,albumin and a 40 kDa protein and a complex of proteinase inhibitorscomposition isolated from plasma consisting essentially of α₂-macroglobulin, a 160 kDa protein, an 80-60 kDa protein and a 20 kDaprotein, b) separating the liquid medium from the leucocytes; c)removing impurities from the liquid medium; and d) recovering from theliquid medium of c) a cytokine composition comprising said transalcomposition and said complex of proteinase inhibitors composition. 8.The method according to claim 7 wherein in step (a) the transalcomposition is added at a final concentration of 6% v/v and the complexof proteinase inhibitors composition is added at a final concentrationof 15 mg/ml.
 9. The method according to claim 8 wherein step a) iscarried out for approximately 10-14 hours at approximately 37.5° C. 10.The method according to claim 7 wherein in step (b) the leucocytes areremoved by centrifugation.
 11. The method according to claim 7 whereinthe impurities comprise human virus, foreign antigens, and antibiotics.12. The method according to claim 11 wherein,a) the human virus isremoved by chemical inactivation with hydrogen peroxide and chloroform;b) the antibiotics are removed by gel filtration; and c) the foreignantigens are removed by negative immunoabsorption on a column containingantibodies specific for the antigens.
 13. A method for producing acytokine composition comprising the steps of:a) culturing leucocytespreviously incubated with a virus in a liquid culture medium containinga transal composition isolated form plasma consisting essentially oftransferrin, albumin and a 40 kDa protein and a complex of proteinaseinhibitors composition isolated from plasma consisting essentially of α₂-macroglobulin, a 160 kDa protein, an 80-60 kDa protein and a 20 kDaprotein; b) removing the leucocytes from the liquid medium; c) treatingthe liquid medium to remove impurities; and d) recovering a cytokinecomposition comprising said transal composition and said complex ofproteinase inhibitors composition; said transal composition beingprepared by a method comprising the steps of: a) mixing plasma withchloroform; b) recovering a first aqueous phase from theplasma/chloroform mixture of step a) and adjusting the pH of the firstaqueous phase to 8.5; c) precipitating gamma globulins from the firstaqueous phase with polyethylene glycol; d) recovering a second aqueousphase from step c); e) passing the second aqueous phase through an anionexchange column equilibrated with 0.01M tris-HCl buffer, pH 8.5 and0.01M NaCl; and f) recovering proteins that bind to the column bypassing a solution of 0.01M tris-HCl buffer, pH 8.5 and 0.5M NaClthrough the column, wherein the proteins eluted in step f) consist ofthe transal composition; and said complex of protease inhibitorscomposition being prepared by a method comprising the steps of: a)mixing plasma with chloroform; b) recovering a first aqueous phase fromthe plasma/chloroform mixture of step a) and adjusting the pH of thefirst aqueous phase to 8.5; c) precipitating gamma globulins from thefirst aqueous phase with polyethylene glycol; d) recovering a secondaqueous phase from step c); e) passing the second aqueous phase throughan anion exchange column equilibrated with 0.01M tris-HCl buffer, pH 8.5and 0.01M NaCl; f) collecting an eluate that passes freely through thecolumn and adjusting the pH of said eluate to pH 6.5; g) passing saideluate through a Cu⁺⁺ chelate anion exchange column equilibrated with0.02M phosphate buffer, pH 6.5 and 0.8M NaCl; and h) recovering proteinsthat bind to the column by eluting said column with 0.1M acetate buffer,pH 4.5 and 0.5M NaCl wherein the proteins eluted in step h) consist ofthe complex of proteinase inhibitors composition.
 14. A cytokinecomposition prepared according to the method of claim
 13. 15. Thecytokine composition of claim 14 further comprising a diluent orcarrier.
 16. A method of enhancing the effectiveness of the immunesystem comprising administering to a person in need of such treatment inan amount effective for said treatment a cytokine composition preparedaccording to claim
 7. 17. The method of claim 16 wherein the persontreated has a viral infection.
 18. The method of claim 16 wherein theperson treated has a bacterial infection.